Caractérisation des événements moléculaires et cellulaires de l’apoptose induite par rbf1, l'homologue de drosophile du gène suppresseur de tumeur rb

The inactivation of the retinoblastoma susceptibility gene (rb) is a preliminary step in the development of many cancers. Consistent with its role of tumor suppressor, pRb inhibits cell proliferation. The role of pRb in apoptosis control is more complex and the molecular mechanisms underlying these functions are poorly described.rbf1 is the rb Drosophila homologue. During my PhD, I characterized the apoptosis pathway induced by Rbf1 in a proliferative tissue. I showed that Rbf1 cooperates with the dE2F2 transcription factor and with the dREAM complex to stimulate the transcription of how gene coding for a RNA binding protein able to induce the degradation of diap1 caspase inhibitor transcripts. Furthermore, Rbf1/dE2F2 proteins repress the transcription of buffy (anti-apoptotic gene of Bcl-2 family) and thus trigger a mitochondrial death pathway dependent of debcl (pro-apoptotic gene of Bcl-2 family). A mitochondrial fragmentation dependent of the Drp1 pro-fission protein promotes the accumulation of reactive oxygen species, which in turn causes JNK pathway activation, leading ultimately to apoptosis. This work clarifies the mechanism of action of Bcl-2 proteins in drosophila. It brings new data about Rbf1 pro-apoptotic function and should provide a better understanding of pRb tumor suppressor activity. Finally, I showed that Rbf1-induced apoptosis leads to compensatory proliferation and defined for the first time specific actors of the JNK pathway involved in this proliferation.L’inactivation du gène de susceptibilité au rétinoblastome (rb) est une étape préalable au développement de nombreux cancers. De façon cohérente avec son rôle de suppresseur de tumeur, pRb inhibe la prolifération cellulaire. Le rôle de pRb dans le contrôle de l’apoptose est plus complexe et les mécanismes moléculaires sous-jacents à ces fonctions sont peu décrits.La drosophile possède un homologue de rb, appelé rbf1. Au cours de ma thèse, j’ai caractérisé la voie d’apoptose induite par Rbf1 dans un tissu prolifératif. J’ai pu montrer que Rbf1 coopère avec le facteur de transcription dE2F2 et le complexe dREAM pour stimuler la transcription du gène how codant pour une protéine de liaison aux ARN capable d’induire la dégradation des transcrits de l’inhibiteur de caspases diap1. Par ailleurs, ces protéines répriment la transcription du gène buffy (gène anti apoptotique de la famille Bcl 2) et ainsi déclenchent une voie de mort mitochondriale dépendante de debcl (gène pro apoptotique de la famille Bcl 2). La fragmentation du réseau mitochondrial, dépendante de la protéine pro-fission Drp1, favorise l’accumulation d’espèces activées de l’oxygène, à l'origine de l'activation de la voie JNK et in fine de l’apoptose. Ces travaux précisent le mode d'action des protéines de la famille Bcl-2 de drosophile. Ils apportent de nouvelles données concernant le rôle pro-apoptotique de Rbf1 et devraient permettre de mieux comprendre l'activité suppresseur de tumeur de pRb. Enfin, j’ai pu montrer que l’apoptose induite par Rbf1 conduit à un mécanisme de prolifération compensatoire et caractériser pour la première fois des acteurs spécifiques de la voie JNK impliqués dans ce mécanisme

Characterisation of molecular and cellular vents of apoptosis induced by rbf1, the drosophila homologue of the rb tumo suppresor gene

L’inactivation du gène de susceptibilité au rétinoblastome (rb) est une étape préalable au développement de nombreux cancers. De façon cohérente avec son rôle de suppresseur de tumeur, pRb inhibe la prolifération cellulaire. Le rôle de pRb dans le contrôle de l’apoptose est plus complexe et les mécanismes moléculaires sous-jacents à ces fonctions sont peu décrits.La drosophile possède un homologue de rb, appelé rbf1. Au cours de ma thèse, j’ai caractérisé la voie d’apoptose induite par Rbf1 dans un tissu prolifératif. J’ai pu montrer que Rbf1 coopère avec le facteur de transcription dE2F2 et le complexe dREAM pour stimuler la transcription du gène how codant pour une protéine de liaison aux ARN capable d’induire la dégradation des transcrits de l’inhibiteur de caspases diap1. Par ailleurs, ces protéines répriment la transcription du gène buffy (gène anti apoptotique de la famille Bcl 2) et ainsi déclenchent une voie de mort mitochondriale dépendante de debcl (gène pro apoptotique de la famille Bcl 2). La fragmentation du réseau mitochondrial, dépendante de la protéine pro-fission Drp1, favorise l’accumulation d’espèces activées de l’oxygène, à l'origine de l'activation de la voie JNK et in fine de l’apoptose. Ces travaux précisent le mode d'action des protéines de la famille Bcl-2 de drosophile. Ils apportent de nouvelles données concernant le rôle pro-apoptotique de Rbf1 et devraient permettre de mieux comprendre l'activité suppresseur de tumeur de pRb. Enfin, j’ai pu montrer que l’apoptose induite par Rbf1 conduit à un mécanisme de prolifération compensatoire et caractériser pour la première fois des acteurs spécifiques de la voie JNK impliqués dans ce mécanisme.The inactivation of the retinoblastoma susceptibility gene (rb) is a preliminary step in the development of many cancers. Consistent with its role of tumor suppressor, pRb inhibits cell proliferation. The role of pRb in apoptosis control is more complex and the molecular mechanisms underlying these functions are poorly described.rbf1 is the rb Drosophila homologue. During my PhD, I characterized the apoptosis pathway induced by Rbf1 in a proliferative tissue. I showed that Rbf1 cooperates with the dE2F2 transcription factor and with the dREAM complex to stimulate the transcription of how gene coding for a RNA binding protein able to induce the degradation of diap1 caspase inhibitor transcripts. Furthermore, Rbf1/dE2F2 proteins repress the transcription of buffy (anti-apoptotic gene of Bcl-2 family) and thus trigger a mitochondrial death pathway dependent of debcl (pro-apoptotic gene of Bcl-2 family). A mitochondrial fragmentation dependent of the Drp1 pro-fission protein promotes the accumulation of reactive oxygen species, which in turn causes JNK pathway activation, leading ultimately to apoptosis. This work clarifies the mechanism of action of Bcl-2 proteins in drosophila. It brings new data about Rbf1 pro-apoptotic function and should provide a better understanding of pRb tumor suppressor activity. Finally, I showed that Rbf1-induced apoptosis leads to compensatory proliferation and defined for the first time specific actors of the JNK pathway involved in this proliferation

Le come-back de la mitochondrie dans l'apoptose chez la drosophile

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Two different specific JNK activators are required to trigger apoptosis or compensatory proliferation in response to Rbf1 in Drosophila

International audienceThe Jun Kinase (JNK) signaling pathway responds to diverse stimuli by appropriate and specific cellular responses such as apoptosis, differentiation or proliferation. The mechanisms that mediate this specificity remain largely unknown. The core of this signaling pathway, composed of a JNK protein and a JNK kinase (JNKK), can be activated by various putative JNKK kinases (JNKKK) which are themselves downstream of different adaptor proteins. A proposed hypothesis is that the JNK pathway specific response lies in the combination of a JNKKK and an adaptor protein upstream of the JNKK. We previously showed that the Drosophila homolog of pRb (Rbf1) and a mutant form of Rbf1 (Rbf1 D253A) have JNK-dependent pro-apoptotic properties. Rbf1 D253A is also able to induce a JNK-dependent abnormal proliferation. Here, we show that Rbf1-induced apoptosis triggers proliferation which depends on the JNK pathway activation. Taking advantage of these phenotypes, we investigated the JNK signaling involved in either Rbf1-induced apoptosis or in proliferation in response to Rbf1-induced apoptosis. We demonstrated that 2 different JNK pathways involving different adaptor proteins and kinases are involved in Rbf1-apoptosis (i.e. Rac1-dTak1-dMekk1-JNK pathway) and in proliferation in response to Rbf1-induced apoptosis (i.e., dTRAF1-Slipper-JNK pathway). Using a transient induction of rbf1, we show that Rbf1-induced apoptosis activates a compensatory proliferation mechanism which also depends on Slipper and dTRAF1. Thus, these 2 proteins seem to be key players of compensatory proliferation in Drosophila

Apoptosis in Drosophila: which role for mitochondria?

International audienceIt is now well established that the mitochon-drion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mam-malian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mito-chondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human

The Drosophila retinoblastoma protein, Rbf1, induces a Debcl-and Drp1-dependent mitochondrial apoptosis

International audienceIn accordance with its tumor suppressor role, the retinoblastoma protein pRb can ensure pro-apoptotic functions. Rbf1, the Drosophila homolog of Rb, also displays a pro-apoptotic activity in proliferative cells. We have previously shown that the Rbf1 pro-apoptotic activity depends on its ability to decrease the level of anti-apoptotic proteins such as the Bcl-2 family protein Buffy. Buffy often acts in an opposite manner to Debcl, the other Drosophila Bcl-2-family protein. Both proteins can localize at the mitochondrion, but the way they control apoptosis still remains unclear. Here, we demonstrate that Debcl and the pro-fission gene Drp1 are necessary downstream of Buffy to trigger a mitochondrial fragmentation during Rbf1-induced apoptosis. Interestingly, Rbf1-induced apoptosis leads to a Debcl-and Drp1-dependent reactive oxygen species production, which in turn activates the Jun Kinase pathway to trigger cell death. Moreover, we show that Debcl and Drp1 can interact and that Buffy inhibits this interaction. Notably, Debcl modulates Drp1 mitochondrial localization during apoptosis. These results provide a mechanism by which Drosophila Bcl-2 family proteins can control apoptosis, and shed light on a link between Rbf1 and mitochondrial dynamics in vivo

Mutating RBF Can Enhance Its Pro-Apoptotic Activity and Uncovers a New Role in Tissue Homeostasis

International audienceThe tumor suppressor retinoblastoma protein (pRb) is inactivated in a wide variety of cancers. While its role during cell cycle is well characterized, little is known about its properties on apoptosis regulation and apoptosis-induced cell responses. pRb shorter forms that can modulate pRB apoptotic properties, resulting from cleavages at caspase specific sites are observed in several cellular contexts. A bioinformatics analysis showed that a putative caspase cleavage site (TELD) is found in the Drosophila homologue of pRb (RBF) at a position similar to the site generating the p76Rb form in mammals. Thus, we generated a punctual mutant form of RBF in which the aspartate of the TELD site is replaced by an alanine. This mutant form, RBF D253A , conserved the JNK-dependent pro-apoptotic properties of RBF but gained the ability of inducing overgrowth phenotypes in adult wings. We show that this overgrowth is a consequence of an abnormal proliferation in wing imaginal discs, which depends on the JNK pathway activation but not on wingless (wg) ectopic expression. These results show for the first time that the TELD site of RBF could be important to control the function of RBF in tissue homeostasis in vivo

Understanding the bactericidal mechanism of Cu(OH)2 nanorods in water through Mg-substitution: high production of toxic hydroxyl radicals by non-soluble particles

International audienceTo date, there is still a lack of definite knowledge regarding the toxicity of Cu(OH)2 nanoparticles towards bacteria. This study was aimed at shedding light on the role played by released cupric ions in the toxicity of nanoparticles. To address this issue, the bactericidal activity of Cu(OH)2 was at first evaluated in sterile water, a medium in which particles are not soluble. In parallel, an isovalent substitution of cupric ions by Mg2+ was attempted in the crystal structure of Cu(OH)2 nanoparticles to increase their solubility and determine the impact on the bactericidal activity. For the first time, mixed Cu1xMgx(OH)2 nanorods (x ≤ 0.1) of about 15 nm in diameter and a few hundred nanometers in length were successfully prepared by a simple co-precipitation at room temperature in mixed alkaline (NaOH/Na2CO3) medium. For E. coli, 100% reduction of one million CFU per mL (6 log10) occurs after only 180 min on contact with both Cu(OH)2 and Cu0.9Mg0.1(OH)2 nanorods. The entire initial inoculum of S. aureus is also killed by Cu(OH)2 after 180 min (100% or 6 log10 reduction), while 0.01% of these bacteria stay alive on contact with Cu0.9Mg0.1(OH)2 (99.99% or 4 log10 reduction). The bactericidal performances of Cu(OH)2 and the magnesium-substituted counterparts (i.e. Cu1xMgx(OH)2) are not linked to cupric ions they release in water since their mass concentrations after 180 min are much lower than minimal concentrations inhibiting the growth of E. coli and S. aureus. Finally, an EPR spin trapping study reveals how these nanorods kill bacteria in water: only the presence of hydrogen peroxide, a by-product of the normal metabolism of oxygen in aerobic bacteria, allows the Cu(OH)2 and its magnesium-substituted counterparts to produce a lethal amount of free radicals, the majority of which are the highly toxic HO•

The drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the b-TrCP homologue slimb

International audienceThe ubiquitin-proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, we identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. We then demonstrated that Uba1 also regulates apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, we show that these apoptotic processes involve the same multimeric E3 ligase-an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of b-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family

Screening of suppressors of bax-induced cell death identifies glycerophosphate oxidase-1 as a mediator of debcl-induced apoptosis in Drosophila

International audienceMembers of the Bcl-2 family are key elements of the apoptotic machinery. In mammals, this multigenic family contains about twenty members, which either promote or inhibit apoptosis. We have previously shown that the mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model. We report here the results of the screening of a P[UAS]-element insertion library performed to identify gene products that modify the phenotypes induced by the expression of bax in Drosophila melanogaster. We isolated 17 putative modifiers involved in various function or process: the ubiquitin/proteasome pathway; cell growth, proliferation and death; pathfinding and cell adhesion; secretion and extracellular signaling; metabolism and oxidative stress. Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways. Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation